Abstract
Studies in the field of developmental biology aim to unravel how a fertilized egg develops into an adult organism and how proteins and other macromolecules work together during this process. With regard to protein function, most of the developmental studies have used genetic and RNA interference approaches, combined with biochemical analyses, to reach this goal. However, there always remains much room for interpretation on how a given protein functions, because proteins work together with many other molecules in complex regulatory networks and it is not easy to reveal the function of one given protein without affecting the networks. Likewise, it has remained difficult to experimentally challenge and/or validate the proposed concepts derived from mutant analyses without tools that directly manipulate protein function in a predictable manner. Recently, synthetic tools based on protein binders such as scFvs, nanobodies, DARPins, and others have been applied in developmental biology to directly manipulate target proteins in a predicted manner. Although such tools would have a great impact in filling the gap of knowledge between mutant phenotypes and protein functions, careful investigations are required when applying functionalized protein binders to fundamental questions in developmental biology. In this review, we first summarize how protein binders have been used in the field, and then reflect on possible guidelines for applying such tools to study protein functions in developmental biology.This article is categorized under:Technologies > Analysis of ProteinsEstablishment of Spatial and Temporal Patterns > GradientsInvertebrate Organogenesis > Flies
Highlights
A key question in developmental biology is how the millions of proteins expressed in each cell function in a highly coordinated manner to properly orchestrate developmental processes
Synthetic tools based on small, high affinity protein binders such as single-chain variable fragments, nanobodies, Design Ankyrin Repeat Proteins (DARPins), affibodies, monobodies, and others have been applied in the field of developmental biology to directly manipulate target proteins in their complex in vivo settings
This method has been used for the visualization of the dynamic distribution of transcription factors with a half-life shorter than the time required for maturation of the GFP DARPin (GFP) fluorophore (>30 min), making it impossible to visualize them by generating direct fluorescent proteins (FPs) fusion proteins
Summary
A key question in developmental biology is how the millions of proteins expressed in each cell function in a highly coordinated manner to properly orchestrate developmental processes. Synthetic tools based on small, high affinity protein binders such as single-chain variable fragments (scFvs), nanobodies, Design Ankyrin Repeat Proteins (DARPins), affibodies, monobodies, and others have been applied in the field of developmental biology to directly manipulate target proteins in their complex in vivo settings By fusing such binders to protein domains of well-characterized properties (functions) and expressing these fusion proteins during development, the properties and the localization of a protein of interest (POI) can be manipulated in a predictable manner. In the LlamaTag approach (Bothma et al, 2018), a ubiquitously distributed cytoplasmic FP is recruited by a nanobody fused to the POI This method has been used for the visualization of the dynamic distribution of transcription factors with a half-life shorter than the time required for maturation of the GFP fluorophore (>30 min), making it impossible to visualize them by generating direct FP fusion proteins. Since the POI does not bind to GFP directly but recruits it via the fused LlamaTag, it is unlikely that the POI is affected by interaction between the protein binder and its target
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